JP2002306190A - Method for separating/recovering poly-3-hydroxyalkanoic acid from biological cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for recovering a poly-3-hydroxyalkanoic acid(PHA), capable of making a separation procedure simple, capable of effectively removing cell-forming components other than the PHA from the biological cells containing the PHA through a process of few steps at a low cost, and capable of obtaining the highly purified PHA in a high yield without having such a fear that chemicals including enzymes, acids, and alkalis may remain in the PHA. SOLUTION: This method for recovering the PHA from the biological cells includes the following processes: a process for crushing the biological cells containing the PHA to give a crushed product; another process for separating the crushed product into a water-soluble fraction and a water-insoluble fraction; and the other process for treating the water-insoluble fraction with an oxidizing agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for recovering poly-3-hydroxyalkanoic acid from living cells containing poly-3-hydroxyalkanoic acid.

[0002]

BACKGROUND ART Microbial polyesters (poly-3-hydroxyalkanoic acids; hereinafter referred to as PHA) typified by poly-3-hydroxybutyric acid (PHB) are different from petroleum-derived synthetic polymers and decomposed by organisms. It has the property that it can be done. Human beings have been using synthetic polymers as plastics and the like for many years, but when those plastics become waste, their property of being difficult to decompose suffers and accumulates at waste disposal sites. Further, performing incineration causes harmful substances such as dioxins and environmental hormones, and causes environmental pollution. On the other hand, microorganism-produced PHA is taken into natural material circulation by being biodegraded, and can be used as a plastic that enables environmental conservation. In addition, there is a possibility that it will be useful as a medical soft member in the future (Japanese Patent Laid-Open No. 5-159).

Hitherto, it has been reported that many bacteria produce and accumulate PHB or other copolymers with hydroxyalkanoic acid in the cells ("Biodegradable Plastic Handbook" (edited by Biodegradable Plastic Research Group)). NTS), p178-197). In particular, Alcaligenes eutrophus strain H16 (Alcaligenes eutr
ophus H16, ATCC No. 17699) and variants thereof have been studied in detail for the production of these polymers,
By changing the carbon source serving as a substrate, a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) or a copolymer containing both units as components is mixed at various ratios. It is disclosed that it is generated (JP-B-6-15604, JP-B-7-14352, JP-B-8-19227, etc.).

[0004] Patent Publication No. 2642937 discloses Pseudomonas oleovorans.
By giving acyclic aliphatic hydrocarbons as a carbon source to ATCC 29347, 3-CC with 6 to 12 carbon atoms can be obtained.
It is disclosed to produce a polyester having hydroxyalkanoic acid (3HA) as a monomer unit.

[0005] Japanese Patent Application Laid-Open No. Hei 5-74492 discloses a genus Methylobacterium and a paracoccus (P.
A method of producing a 3HB and 3HV copolymerized polyester by contacting a bacterium belonging to the genus Aracoccus, Alcaligenes or Pseudomonas with a primary alcohol having 3 to 7 carbon atoms is disclosed.

[0006] JP-A-5-93049 and JP-A-7-265065 disclose Aeromonas caviae.
(Aeromonas Caviae) is disclosed to produce a two-component copolyester of 3HB and 3-hydroxyhexanoic acid (3HHx) by culturing oleic acid or olive oil as a carbon source.

[0007] Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans I
It is disclosed that FO 13852 strain produces a polyester having 3HB and 4-hydroxybutyric acid (4HB) as monomer units by culturing using gluconic acid and 1,4-butanediol as carbon sources.

[0008] Such PHA produced and accumulated by microorganisms is generally extracted from cells using an organic solvent in which PHA is dissolved, specifically, a chlorine-based organic solvent such as chloroform or dichloromethane. . However, when large-scale production is considered, in order to separate PHA from microbial cells, the above-mentioned method uses a large amount of a chlorine-based organic solvent, which is not practical.

[0009] From such a viewpoint, various methods have been studied for separating PHA from living cells such as microbial cells without using an organic solvent.

Japanese Patent Application Laid-Open No. 57-174094 discloses P
A method for separating PHA from microbial cells by crushing the cells by heating and pressurizing the HA-accumulating cells and releasing the pressure is disclosed.

US Pat. No. 4,562,245 and JP-A-59-205992 disclose a method of separating PHB by digesting PHB-containing cells with a proteolytic enzyme composition.

US Pat. No. 4,910,145 and JP-A-60-145097 disclose a method of separating PHB from cells using a solubilizing agent for cell components such as a protease and a surfactant. .

JP-A-63-226291 discloses a method of converting cells into spheroplasts, crushing them by sonic vibration treatment, and separating the uppermost PHA formed after centrifugation. Is described.

JP-A-5-336982 discloses that PH
A method of dissolving the cytoplasm of the A-accumulating microorganism with a protease and purifying the polymer and / or copolymer using a surfactant is disclosed.

JP-A-7-31487, JP-A-7-3
1488 and JP-A-7-31489 disclose P
Disclosed are a method of treating HB-containing cells with an alkali to separate PHB, a method of treating PHB at a high temperature and a high pressure to separate PHB, and a method of combining both to separate PHB.

Japanese Patent Publication No. Hei 8-508888 discloses a PH.
A method of separating PHA from cells by treating the A-accumulating cells with a protease, treating the cells with an appropriate chelating agent, and further treating with hydrogen peroxide is disclosed.

[0017]

However, in the above-mentioned treatment, since the operation is complicated and there is a possibility of remaining using an enzyme, an acid or an alkali, it is possible to recover PHA with higher simplicity and high purity. There was a need for a way to get it.

An object of the present invention is to solve the above-described problems in the prior art, to efficiently remove cell components other than PHA from PHA-containing living cells at a low number of steps and at low cost, and to obtain high-purity PHA. PH for high yield
A method for recovering A.

In the present invention, "biological cells"
It includes not only microbial cells, but also cells of higher organisms such as plant cells that can produce PHA by incorporating a PHA synthesis gene.

[0020]

DISCLOSURE OF THE INVENTION The present inventors have proposed a method for efficiently removing cell constituents other than PHA from PHA-containing living cells at low cost with a small number of steps and obtaining PHA with high purity in high yield. As a result of diligent studies on the method for recovering PHA, the following invention was reached.

That is, the present invention provides a method for recovering poly-3-hydroxyalkanoic acid from living cells, the step of crushing living cells containing poly-3-hydroxyalkanoic acid to obtain a crushed product, Separating water into a water-soluble fraction and a water-insoluble fraction, and treating the water-insoluble fraction with an oxidizing agent.
The present invention relates to a method for recovering hydroxyalkanoic acid.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION In the step of crushing living cells in the present invention, ultrasonic crushing, homogenizer, pressure crushing, bead impact, crushing, and crushing (such as glass powder and alumina powder) are used. It is desirable to use a method that does not use a drug, such as a method of adding a drug and grinding in a mortar), a freeze-thawing method, and the like. Further, the solid component and the soluble component are separated from the resuspension by a method such as centrifugation, and the solid component containing the PHA component is treated with a peroxide compound.

The oxidizing agent used in the method of the present invention is a peroxide compound or sodium hypochlorite.

The peroxide compound used in the method of the present invention includes hydrogen peroxide, organic peracids such as perbenzoic acid, metachloroperbenzoic acid, formic acid, peracetic acid, monoperoxyphthalic acid and trifluoroperacetic acid, and esters thereof. However, it is preferable to use either hydrogen peroxide or sodium percarbonate because it can be easily used as an aqueous solution and the reaction is relatively mild. Hydrogen peroxide is very effective from the viewpoint of increasing the purity of the recovered PHA without any residue after the treatment. However, depending on the unit structure of the PHA, there is a possibility that the structure may change due to oxidation. It is desirable to use sodium percarbonate in which a milder reaction proceeds.

The concentration of hydrogen peroxide used in the method of the present invention is
Preferably, it is in the range of 3.0% to 31.0% and the concentration of sodium percarbonate is in the range of 5.0% to 20.0%.

It is desirable that the treatment temperature of these peroxide compounds is in the range of 80 ° C. to 100 ° C. If it is lower than that, the effect is low and the purity of the recovered material is low. There is. The reaction time is preferably from 30 minutes to 2 hours, more preferably about 1 hour.

After the treatment, washing with water is performed,
It is necessary to repeat stirring and centrifugation about a degree. If residual trace metals are not desired, water may be deionized, distilled,
It is desirable to use purified water such as pure water. When sodium percarbonate is used as the peroxide compound, care must be taken so that sodium percarbonate does not remain. When performing centrifugation, it is important to pay special attention to the temperature, from 0 ° C to 1 ° C.
It is desirable to carry out at about 0 ° C, preferably at 0 ° C to 5 ° C.

The concentration of sodium hypochlorite used in the method of the present invention is preferably about 4% (effective chlorine concentration of about 1.7%) to about 6% (effective chlorine concentration of about 2.5%), and the treatment temperature is 0 ° C. to 10 ° C. It is desirable that In this case, as in the case of using a peroxide compound, it is necessary to wash with water after the treatment and repeat the stirring and centrifugation about twice.

When it is necessary to obtain high-purity PHA, it is possible to carry out, for example, an enzyme, an oxidizing agent, a surfactant or a chemical treatment using a combination thereof after the PHA separation and washing operations.

The living cells to which the method of the present invention can be used may be any living cells as long as they accumulate PHA in the body.
The present invention can also be applied to plant cells in which a production gene has been modified.

Next, the present invention will be described in more detail by way of examples.

The M9 medium used in each Example has the following composition. M9 medium composition (in 1 liter): Na ₂ HPO ₄ 6.2 g KH ₂ PO ₄ 3.0 g NaCl 0.5 g NH ₄ Cl 1.0 g water balance (pH 7.0) The microorganism used in this example was Ralstonia eutropha TB64 strain (Ralstoniaeutropha TB64, JP 2000-
No. 166587; deposited at the National Institute of Advanced Industrial Science and Technology (AIST) (Deposit No .: FER
M BP-6933)), and Pseudomonas oleovorans
(Pseudomonas oleovorans) ATCC 29347 (American
(Sold by Type Culture Collection).

[0033]

EXAMPLES [Example 1] A colony of the TB64 strain on M9 agar medium containing 0.1% sodium malate was inoculated into 50 mL of M9 medium containing 0.5% sodium malate in a 500 mL shake flask. And cultured with shaking at 30 ° C. Two
Four hours later, 5 mL of the culture solution was added to only NH ₄ Cl as a nitrogen source for 1 hour.
Sodium malate 0.5% in M9 medium adjusted to / 10 concentration
% Of the medium was added to the medium and shaken in the same manner to accumulate PHB in the cells. After 48 hours, the PHB-accumulating cells were harvested by centrifugation, resuspended in 40 mL of distilled water and divided into four equal portions (10 mL each). These were numbered from 1 to 4, and the following processing was performed.

1: Control: Washed with methanol after further centrifugation, freeze-dried, weighed, and then chloroform
An extraction operation was performed for a period of time, and the extract was filtered, concentrated, reprecipitated with methanol, and dried under reduced pressure to obtain a control polymer.

2: 40 mL of 31% hydrogen peroxide solution (manufactured by Mitsubishi Gas Chemical Company; JIS K-8230) was added, and the mixture was treated at 80 ° C. for 1 hour.

3: Make the suspension up to 50 mL with distilled water, and use a French press (Otake Manufacturing Co., Ltd .: French Press)
5501), and centrifuged at 4 ° C. and 29400 m / s ² (3000 G) for 30 minutes. Then add another 40 mL of distilled water,
4 ° C., washed by centrifugation for 30 minutes at 29400m / s ² (3000G).

4: After performing the same operation as in 3, the precipitated portion was suspended in 10 mL of distilled water, and 31% aqueous hydrogen peroxide was added to 4 mL of distilled water.
0 mL was added and the treatment was performed at 80 ° C. for 1 hour.

5: After performing the same operation as in 3, the precipitated portion was suspended in 10 mL of distilled water, and 5 mL of sodium hypochlorite solution (manufactured by Kishida Chemical Co .; sodium hypochlorite)
12% (contains an effective chlorine concentration of 5% or more).
Time processing was performed.

Numbers 2 to 4 are obtained by centrifugation (4 ° C., 29400 m
/ s ² (3000 G) for 30 minutes), cooled to 4 ° C. again, added 40 mL of distilled water, stirred well, centrifuged twice under the same conditions, freeze-dried the precipitate, and weighed.

The following operations were performed to evaluate the "recovery rate" and "purity" shown below.

Chloroform (30 mL) was added to the freeze-dried samples 1 to 5, and the mixture was stirred and extracted at 60 ° C. for 24 hours. 0.45 chloroform solution from which PHB was extracted
Filtered with a PTFE filter of μm, concentrated by a rotary evaporator, poured into 10 times the volume of methanol,
B was precipitated and recovered. These were dried under reduced pressure and weighed.

The mass ratio of PHB obtained by chloroform extraction of 2 to 5 samples to 1, which is a control, is referred to as “recovery ratio”, and PHB of PHB obtained by chloroform extraction of each sample with respect to the sample before chloroform extraction. Table 1 shows the mass ratio as “purity”.

[0043]

[Table 1]

The recovery rate did not change much, and the purity was the sample that had been subjected to French press treatment and subsequent hydrogen peroxide treatment.
(4) and the sample (5) treated with hypochlorous acid were good.

The obtained PHB was subjected to gel permeation chromatography (GPC; Tosoh HLC-8020, column: Polymer Laboratory PLgelMIXED-C (5 μm).
m), solvent: chloroform, converted to polystyrene). Table 2 shows the results.

[0046]

[Table 2]

Almost no difference in the molecular weight of each sample was observed.

Example 2 M containing 0.1% of n-nonanoic acid
A colony of Pseudomonas oleovorans on 9 agar medium was inoculated into 50 mL of M9 medium containing 0.3% of n-nonanoic acid, and cultured with shaking at 30 ° C. 40 hours later, culture medium 5m
L was added to 1 L of M9 medium containing 0.1% of n-nonanoic acid and 0.1% of 5-phenylvaleric acid, followed by shaking culture. After an additional 40 hours, the cells were harvested by centrifugation and the nitrogen source N
The cells were resuspended in 1 L of M9 medium containing 0.1% of n-nonanoic acid and 0.1% of 5-phenylvaleric acid without containing H ₄ Cl, and the cells were shaken in the same manner to give 3-hydroxynonanoic acid and 3-hydroxyvalic acid. PHA containing heptanoic acid, 3-hydroxyvaleric acid, and 3-hydroxy-5-phenoxyvaleric acid as units were accumulated. After 40 hours, the PHA-accumulating cells were harvested by centrifugation and subjected to the following treatment.

6: Control: Washed again with methanol after centrifugation, freeze-dried and weighed, then chloroform
An extraction operation was performed for a period of time, and the extract was filtered, concentrated, reprecipitated with methanol, and dried under reduced pressure to obtain a control polymer.

7: 40 mL of 31% aqueous hydrogen peroxide (manufactured by Mitsubishi Gas Chemical Company; JIS K-8230) was added, and the mixture was treated at 80 ° C. for 1 hour.

[0051] 8: suspension female and up to 50mL with distilled water, a French press (Ohtake Seisakusho Co., Ltd.: French press 5501) after, 4 ℃, in the 29400m / s ² (3000G) 30
Centrifugation was performed for minutes. Thereafter, 40 mL of distilled water was further added, and centrifugation was performed at 4 ° C. and 29400 m / s ² (3000 G) for 30 minutes for washing.

9: After performing the same operation as in 3, the precipitated portion was suspended in 10 mL of distilled water, and 31% aqueous hydrogen peroxide was added to 4 mL of distilled water.
0 mL was added and the treatment was performed at 80 ° C. for 1 hour.

The numbers 7 to 9 are obtained by centrifugation (4 ° C., 29400 m
/ s ² (3000 G) for 30 minutes), and again added 40 mL of post-distilled water, stirred well, centrifuged twice under the same conditions, freeze-dried the precipitate, and weighed it.

These samples were extracted with chloroform in the same manner as in Example 1, and the recovery and purity were determined. Table 3 shows the results.

[0055]

[Table 3]

The recovery rate was not significantly changed, and the purity was the same as that of the sample subjected to the French press treatment and the subsequent hydrogen peroxide treatment.
(9) was the best.

The obtained PHB was subjected to gel permeation chromatography (GPC; Tosoh HLC-8020, column: Polymer Laboratory PLgelMIXED-C (5 μm).
m), solvent: chloroform, converted to polystyrene). The results are shown in Table 4.

[0058]

[Table 4]

Almost no difference in the molecular weight of each sample was observed.

[0060]

Industrial Applicability According to the method of the present invention, polyhydroxyalkanoic acid accumulated in living cells such as microorganisms can be recovered at a high recovery rate with a simple method and while substantially maintaining the original molecular weight. It has become possible.

Continuing on the front page (72) Inventor Tomohiro Suzuki 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Tsutomu Honma 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Takeshi Nomoto 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Etsuko Sugawa 3-30-2 Shimomaruko 3-chome, Ota-ku, Tokyo Inside Canon Inc. (72) Invention Person Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo F-term in Canon Inc. (reference) 4B064 AD83 CA01 CA02 CE02 CE20 DA01 DA16

Claims (10)

[Claims] 1. A method for recovering poly-3-hydroxyalkanoic acid from living cells, comprising the steps of: crushing living cells containing poly-3-hydroxyalkanoic acid to obtain a crushed product; A method for recovering poly-3-hydroxyalkanoic acid from living cells, comprising: a step of separating a water-insoluble fraction into a water-insoluble fraction; and a step of treating the water-insoluble fraction with an oxidizing agent. 2. The method according to claim 1, wherein the step of crushing the living cells is performed by any one of an ultrasonic crushing method, a homogenizer method, a pressure crushing method, a bead impact method, a grinding method, a crushing method, and a freeze-thawing method. The described method. 3. The method according to claim 1, wherein said oxidizing agent is a peroxide compound.
Or the method of 2. 4. The method according to claim 3, wherein the peroxide compound is one of hydrogen peroxide and sodium percarbonate. 5. The method according to claim 1, wherein the concentration of the hydrogen peroxide in the oxidizing agent is 3.
5. The method of claim 4, wherein the range is from 0% to 31.0%. 6. The method of claim 4, wherein the concentration of said sodium percarbonate in said oxidizing agent ranges from 5.0% to 20.0%. 7. The method according to claim 5, wherein the treatment temperature of the treatment with the oxidizing agent is in the range of 80 ° C. to 100 ° C. 8. The method according to claim 1, wherein the oxidizing agent is sodium hypochlorite. 9. The method according to claim 8, wherein the concentration of the sodium hypochlorite in the oxidizing agent is from 4% (effective chlorine concentration of about 1.7%) to 6% (effective chlorine concentration of about 2.5%). The described method. 10. The method according to claim 9, wherein the treatment temperature of the treatment with sodium hypochlorite is 0 ° C. to 10 ° C.